Transgenic Cells Expressing Glucosyltransferase Nucleic Acids

ABSTRACT

The invention relates to transgenic cells which have been transformed with nucleic acids encoding glycosyltransferase polypeptides (GTases) and vectors for use in transformation of said cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 10/203,319filed Aug. 9, 2002, which is a 371 national stage entry of internationalpatent application no. PCT/GB2001/00477, filed Feb. 8, 2001, designatingthe United States of America, and published in United Kingdom on Aug.16, 2001, as WO 2001/59140, the entire disclosure of which isincorporated herein by reference. Priority is claimed based on UnitedKingdom patent application no. 0002814.2, filed Feb. 9, 2000.

BACKGROUND OF THE INVENTION

The invention relates to transgenic cells which have been transformedwith glucosyltransferase (GTases) nucleic acids.

GTases are enzymes which post-translationally transfer glucosyl residuesfrom an activated nucleotide sugar to monomeric and polymeric acceptormolecules such as other sugars, proteins, lipids and other organicsubstrates. These glucosylated molecules take part in diverse metabolicpathways and processes. The transfer of a glucosyl moiety can alter theacceptor's bioactivity, solubility and transport properties within thecell and throughout the plant. One family of GTases in higher plants isdefined by the presence of a C-terminal consensus sequence. The GTasesof this family function in the cytosol of plant cells and catalyse thetransfer of glucose to small molecular weight substrates, such asphenylpropanoid derivatives, coumarins, flavonoids, other secondarymetabolites and molecules known to act as plant hormones. Availableevidence indicates that GTases enzymes can be highly specific, such asthe maize and Arabidopsis GTases that glucosylate indole-3-acetic acid(IAA).

The production and use of paper has increased in the last 10 years. Forexample, between 1989 and 1999 the production of paper and board in theUK has increased from 4.6 to 6.6 million tonnes. Worldwide consumptionhas also reflected a general increase in paper usage. For example, inthe UK per capita consumption of paper is over 200 kg per annum. In theUSA this figure is over 300 kg per annum.

Wood used in the paper industry is initially particulated, typically bychipping, before conversion to a pulp which can be utilised to producepaper. The pulping process involves the removal of lignin. Lignin is amajor non-carbohydrate component of wood and comprises approximately onequarter of the raw material in wood pulp. The removal of lignin isdesirable since the quality of the paper produced from the pulp islargely determined by the lignin content. Many methods have beendeveloped to efficiently and cost effectively remove lignin from woodpulp. These methods can be chemical, mechanical or biological. Forexample, chemical methods to pulp wood are disclosed in WO9811294,EP0957198 and WO0047812. Although chemical methods are efficient meansto remove lignin from pulp it is known that chemical treatments canresult in degradation of polysaccharides and is expensive. Moreover, toremove residual lignin from pulp it is necessary to use strong bleachingagents which require removal before the pulp can be converted intopaper. These agents are also damaging to the environment.

Biological methods to remove lignin are known. There are howeverdisadvantages associated with such methods. For example it is importantto provide micro-organisms (eg bacteria and/or fungi) which only secreteligninolytic enzymes which do not affect cellulose fibres. This methodis also very time consuming (can take 3-4 weeks) and expensive due tothe need to provide bioreactors. Biological treatment can also includepre-treatment of wood chips to make them more susceptible to furtherbiological or chemical pulping.

It is therefore desirable to provide further means by which lignin canbe efficiently and cost effectively removed from wood pulp which do nothave the disadvantages associated with prior art methods.

For the sake of clarity reference herein to transgenic means a plantwhich has been genetically modified to include a nucleic acid sequencenot naturally found in said plant. For example, by over-expression ofmonolignol glucosyltransferases in planta, plant cell wall propertiesmay be altered through increasing the flux through biosyntheticintermediates that are obligatory for incorporation and assembly of thelignin polymer. Conversely, reduction of the monolignol glucoside pools,such as through the use of nucleic acid comprising GTase sequences inantisense configuration may lead to altered properties through reducingthe flux through specific intermediates. Changes in lignin composition,such as with decreased ratios of coniferyl alcohol to sinapyl alcoholare highly desirable in paper and pulping processes, because the morehighly methylated lignin (sinapyl alcohol) is more easily removed duringpulping processes (Chiang et al (1988) TAPPI J. 71, 173-176).

In some applications it may be desirable to change lignin compositionand increase the lignin content of a plant cell to increase themechanical strength of wood. This would have utility in, for example theconstruction industry or in furniture making.

Both lignin content and the level of cross-linking of polysaccharidepolymers within plant cell walls, also play an important role indetermining texture and quality of raw materials through altering thecell walls and tissue mechanical properties. For example, there isconsiderable interest in reducing cell separation in edible tissuessince this would prevent over-softening and loss of juiciness.Phenolics, such as ferulic acid, play an important role in cell adhesionsince they can be esterified to cell wall polysaccharides duringsynthesis and oxidatively cross-linked in the wall, thereby increasingrigidity. Most non-lignified tissues contain these phenolic componentsand their levels can be modified by altering flux through the samemetabolic pathways as those culminating in lignin. Therefore, in thesame way as for the manipulation of lignin composition and content,GTase nucleic acid in sense and/or antisense configurations can be usedto affect levels of ferulic acid and related phenylpropanoid derivativesthat function in oxidative cross-linking. These changes in content haveutility in the control of raw material quality of edible plant tissues.

Lignin and oxidative cross-linking in plant cell walls also playimportant roles in stress and defence responses of most plant species.For example, when non-woody tissues are challenged by pests or pathogenattack, or suffer abiotic stress such as through mechanical damage or UVradiation, the plant responds by localised and systemic alteration incell wall and cytosolic properties, including changes in lignin contentand composition and changes in cross-linking of other wall components.Therefore, it can also be anticipated that cell- or tissue-specificchanges in these responses brought about by changed levels of therelevant GTase activities will have utility in protecting the plant tobiotic attack and biotic/abiotic stresses.

GTases also have utility with respect to the modification ofantioxidants. Reactive oxygen species are produced in all aerobicorganisms during respiration and normally exist in a cell in balancewith biochemical anti-oxidants. Environmental challenges, such as bypollutants, oxidants, toxicants, heavy metals and so on, can lead toexcess reactive oxygen species which perturb the cellular redox balance,potentially leading to wide-ranging pathological conditions. In animalsand humans, cardiovascular diseases, cancers, inflammatory anddegenerative disorders are linked to events arising from oxidativedamage.

Because of the current prevalence of these diseases, there isconsiderable interest in anti-oxidants, consumed in the diet or appliedtopically such as in UV-screens. Anti-oxidant micronutrients obtainedfrom vegetables and fruits, teas, herbs and medicinal plants are thoughtto provide significant protection against health problems arising fromoxidative stress. Well known anti-oxidants from plant tissues includefor example: quercetin, luteolin, and the catechin, epicatechin andcyanidin groups of compounds.

Caffeic acid (3,4-dihydroxycinnamic acid) is a further example of ananti-oxidant with beneficial therapeutic properties.

Certain plant species, organs and tissues are known to have relativelyhigh levels of one or more compounds with anti-oxidant activity. Greateraccumulation of these compounds in those species, their widerdistribution in crop plants and plant parts already used for food anddrink production, and the increased bioavailability of anti-oxidants(absorption, metabolic conversions and excretion rate) are threefeatures considered to be highly desirable.

It will be apparent that changed levels of the relevant GTase activitiescapable of glucosylating anti-oxidant compounds in planta will allow theproduction of anti-oxidants with beneficial properties. GTase sequencescan also be expressed in prokaryotes or simple eukaryotes, such asyeast, to produce enzymes for biotransformations in those cells, or asin vitro processing systems.

SUMMARY OF THE INVENTION Statements of Invention

According to an aspect of the invention there is provided a transgeniccell comprising a nucleic acid molecule which encodes a polypeptidewhich has:

-   i) glucosyltransferase activity:-   ii) is selected from the group comprising sequences of FIGS. 1A, 2A,    3A, 4A, 5A, 6A, 7A, 8A, 9A, 10A, 11A, 12, 13, 14, 15, 16, 17, 18,    19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32-   iii) nucleic acids which hybridise to the sequences represented    in (ii) above; and-   iv) nucleic acid sequences which are degenerate as a result of the    genetic code to the sequences defined in (i) and (ii) above.

In a further preferred embodiment of the invention said nucleic acidmolecule anneals under stringent hybridisation conditions to thesequence presented in FIGS. 1A, 2A, 3A, 4A, 5A, 6A, 7A, 8A, 9A, 10A,11A, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32

More preferably still said nucleic acid molecule is selected from FIGS.7A, 8A, 9A, 10A, 15, 18, 19, 28 or 31.

Stringent hybridisation/washing conditions are well known in the art.For example, nucleic acid hybrids that are stable after washing in0.1×SSC, 0.1% SDS at 60° C. It is well known in the art that optimalhybridisation conditions can be calculated if the sequence of thenucleic acid is known. For example, hybridisation conditions can bedetermined by the GC content of the nucleic acid subject tohybridisation. Please see Sambrook et al (1989) Molecular Cloning; ALaboratory Approach. A common formula for calculating the stringencyconditions required to achieve hybridisation between nucleic acidmolecules of a specified homology is:

T _(m)=81.5° C.+16.6 Log [Na⁺]+0.41[% G+C]−0.63 (% formamide).

In a preferred embodiment of the invention said transgenic cell is aeukaryotic cell. Preferably said eukaryotic cell is a plant cell oryeast cell.

In an alternative embodiment of the invention said transgenic cell is aprokaryotic cell.

In a further preferred embodiment of the invention the nucleic acidmolecule is selected from the group comprising: antisense sequences ofthe sequences of any one of FIGS. 1C, 2C, 3C, 4C, 5C, 6C, 7C, 8C, 9C,10C and 11C or parts thereof, or antisense sequences of the sensesequences presented in FIGS. 12-32. More preferably still said antisensesequence is selected from FIG. 7C or 9C

In a further preferred embodiment of the invention said nucleic acid iscDNA.

In a yet further preferred embodiment of the invention said nucleic acidis genomic DNA.

In yet still a further preferred embodiment of the invention said plantis a woody plant selected from: poplar; eucalyptus; Douglas fir; pine;walnut; ash; birch; oak; teak; spruce. Preferably said woody plant is aplant used typically in the paper industry, for example poplar.

Methods to transform woody species of plant are well known in the art.For example the transformation of poplar is disclosed in U.S. Pat. No.4,795,855 and WO9118094. The transformation of eucalyptus is disclosedin EP1050209 and WO9725434. Each of these patents is incorporated intheir entirety by reference.

In a still further preferred embodiment of the invention said plant isselected from: corn (Zea mays), canola (Brassica napus, Brassica rapassp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secalecerale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower(helianthus annuas), wheat (Tritium aestivum), soybean (Glycine max),tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Iopmoeabatatus), cassaya (Manihot esculenta), coffee (Cofea spp.), coconut(Cocos nucifera), pineapple (Anana comosus), citris tree (Citrus spp.)cocoa (Theobroma cacao), tea (Camellia senensis), banana (Musa spp.),avacado (Persea americana), fig (Ficus casica), guava (Psidium guajava),mango (Mangifer indica), olive (Olea europaea), papaya (Carica papaya),cashew (Anacardium occidentale), macadamia (Macadamia intergrifolia),almond (Prunus amygdalus), sugar beets (Beta vulgaris), oats, barley,vegetables and ornamentals.

Preferably, plants of the present invention are crop plants (forexample, cereals and pulses, maize, wheat, potatoes, tapioca, rice,sorghum, millet, cassaya, barley, pea, and other root, tuber or seedcrops. Important seed crops are oil-seed rape, sugar beet, maize,sunflower, soybean, and sorghum. Horticultural plants to which thepresent invention may be applied may include lettuce, endive, andvegetable brassicas including cabbage, broccoli, and cauliflower, andcarnations and geraniums. The present invention may be applied intobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper,chrysanthemum.

Grain plants that provide seeds of interest include oil-seed plants andleguminous plants. Seeds of interest include grain seeds, such as corn,wheat, barley, rice, sorghum, rye, etc. Oil-seed plants include cotton,soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut,etc. Leguminous plants include beans and peas. Beans include guar,locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, limabean, fava been, lentils, chickpea, etc.

According to a further aspect of the invention there is provided avector comprising the nucleic acid according to the invention operablylinked to a promoter.

“Vector” includes, inter alia, any plasmid, cosmid, phage orAgrobacterium binary vector in double or single stranded linear orcircular form which may or may not be self-transmissable or mobilizable,and which can transform a prokaryotic or eukaryotic host either byintegration into the cellular genome or exist extrachromosomally (e.g.autonomous replicating plasmid with an origin of replication ie anepisomal vector).

Suitable vectors can constructed, containing appropriate regulatorysequences, including promoter sequences, terminator fragments,polyadenylation sequences, enhancer sequences, marker genes and othersequences as appropriate. For further details see, for example,Molecular Cloning: Laboratory Manual: 2^(nd) edition, Sambrook et al.1989, Cold Spring Habor Laboratory Press or Current Protocols inMolecular Biology, Second Edition, Ausubel et al. Eds., John Wiley &Sons, 1992.

Specifically included are shuffle vectors by which is meant a DNAvehicle capable, naturally or by design, of replication in two differenthost organisms, which may be selected from actinomycetes and relatedspecies, bacteria and eukaryotic (e.g. higher plant, mammalian, yeast orfungal cells).

A vector including nucleic acid according to the invention need notinclude a promoter or other regulatory sequence, particularly if thevector is to be used to introduce the nucleic acid into cells forrecombination into the gene.

Preferably the nucleic acid in the vector is under the control of, andoperably linked to, an appropriate promoter or other regulatory elementsfor transcription in a host cell such as a microbial, (e.g. bacterial),or plant cell. The vector may be a bi-functional expression vector whichfunctions in multiple hosts. In the case of GTase genomic DNA this maycontain its own promoter or other regulatory elements and in the case ofcDNA this may be under the control of an appropriate promoter or otherregulatory elements for expression in the host cell.

By “promoter” is meant a nucleotide sequence upstream from thetranscriptional initiation site and which contains all the regulatoryregions required for transcription. Suitable promoters includeconstitutive, tissue-specific, inducible, developmental or otherpromoters for expression in plant cells comprised in plants depending ondesign. Such promoters include viral, fungal, bacterial, animal andplant-derived promoters capable of functioning in plant cells.

Constitutive promoters include, for example CaMV 35S promoter (Odell etal. (1985) Nature 313, 9810-812); rice actin (McElroy et al. (1990)Plant Cell 2: 163-171); ubiquitin (Christian et al. (1989) Plant Mol.Biol. 18 (675-689); pEMU (Last et al. (1991) Theor Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3. 2723-2730); ALS promoter(U.S. Application Seriel No. 08/409,297), and the like. Otherconstitutive promoters include those in U.S. Pat. Nos. 5,608,149;5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680, 5,268,463; and5,608,142.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducedgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425 andMcNellis et al. (1998) Plant J. 14(2): 247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227: 229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156, herein incorporated by reference.

Where enhanced expression in particular tissues is desired,tissue-specific promoters can be utilised. Tissue-specific promotersinclude those described by Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7): 792-803;Hansen et al. (1997) Mol. Gen. Genet. 254(3): 337-343; Russell et al.(1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) PlantPhysiol. 112(3): 1331-1341; Van Camp et al. (1996) Plant Physiol.112(2): 525-535; Canevascni et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5): 773-778; Lam(1994) Results Probl. Cell Differ. 20: 181-196; Orozco et al. (1993)Plant Mol. Biol. 23(6): 1129-1138; Mutsuoka et al. (1993) Proc. Natl.Acad. Sci. USA 90 (20): 9586-9590; and Guevara-Garcia et al (1993) PlantJ. 4(3): 495-50.

“Operably linked” means joined as part of the same nucleic acidmolecule, suitably positioned and oriented for transcription to beinitiated from the promoter. DNA operably linked to a promoter is “undertranscriptional initiation regulation” of the promoter. In a preferredaspect, the promoter is an inducible promoter or a developmentallyregulated promoter.

Particular of interest in the present context are nucleic acidconstructs which operate as plant vectors. Specific procedures andvectors previously used with wide success upon plants are described byGuerineau and Mullineaux (1993) (Plant transformation and expressionvectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOSScientific Publishers, pp 121-148. Suitable vectors may include plantviral-derived vectors (see e.g. EP-A-194809).

If desired, selectable genetic markers may be included in the construct,such as those that confer selectable phenotypes such as resistance toantibodies or herbicides (e.g. kanamycin, hygromycin, phosphinotricin,chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinonesand glyphosate).

According to a further aspect of the invention there is provided amethod of enhancing monolignol glucoside synthesis in a plant comprisingcausing or allowing expression of at least one GTase nucleic acidaccording to the invention in a plant. Preferably the plant is a woodyplant species.

According to a further aspect of the invention there is provided amethod of inhibiting monolignol glucoside synthesis in a plantcomprising causing or allowing expression of at least one GTaseantisense nucleic acid according to the invention in a plant. Preferablythe plant is a woody plant species.

Inhibition of GTase expression may, for instance, be achieved usinganti-sense technology.

In using anti-sense genes or partial gene sequences to down-regulategene expression, a nucleotide sequence is placed under the control of apromoter in a “reverse orientation” such that transcription yields RNAwhich is complementary to normal mRNA transcribed from the “sense”strand of the target gene. See, for example, Rothstein et al, 1987;Smith et al, (1998), Nature 334, 724-726; Zhang et al (1992) The PlantCell 4, 1575-1588, English et al. (1996) The Plant Cell 8, 179 188.Antisense technology is also reviewed in Bourque (1995), Plant Science105, 125-149, and Flavell (1994) PNAS USA 91, 3490-3496.

According to a further aspect of the invention there is provided anucleotide sequence encoding an antisense RNA molecule complementary toa sense mRNA molecule encoding for a polypeptide having a glucosyltransferase activity in the biosynthesis of at least a monolignolglucoside in lignin biosynthesis in a plant, which nucleotide sequenceis under transcriptional control of a promoter and a terminator, bothpromoter and terminator capable of functioning in plant cells.

Suitable promoters and terminators are referred to hereinabove.

According to a further aspect of the invention there is provided anucleotide sequence according to the invention comprising atranscriptional regulatory sequence, a sequence under thetranscriptional control thereof which encodes an RNA which consists of aplurality of subsequences, characterised in that the RNA subsequencesare antisense RNAs to mRNAs of proteins having a GTase activity in thelignin biosynthesis pathway in plant cells.

In particular, the said RNA subsequences are antisense RNAs to mRNAs ofGTase having a GTase activity in the lignin biosynthesis pathway inplant cells, such as the GTase of FIGS. 1-11(C)

The nucleotide sequence may encode an RNA having any number ofsubsequences. Preferably, the number of subsequences lies between 2 and7 (inclusive) and more preferably lies between 2-4.

According to a further aspect of the invention there is provided a hostcell transformed with nucleic acid or a vector according to theinvention, preferably a plant or a microbial cell. The microbial cellmay be prokaryotic (eg Escherchia coli, Bacillus subtilis) or eukaryotic(eg Saccharomyces cerevisiae).

In the transgenic plant cell the transgene may be on an extra-genomicvector or incorporated, preferably stably, into the genome. There may bemore than one heterologous nucleotide sequence per haploid genome.

According to a yet further aspect of the invention there is provided amethod of transforming a plant cell comprising introduction of a vectorinto a plant cell and causing or allowing recombination between thevector and the plant cell genome to introduce a nucleic acid accordingto the invention into the genome.

Plants transformed with a DNA construct of the invention may be producedby standard techniques known in the art for the genetic manipulation ofplants. DNA can be introduced into plant cells using any suitabletechnology, such as a disarmed Ti-plasmid vector carried byAgrobacterium exploiting its natural gene transferability (EP-A-270355,EP-A-0116718, NAR 12(22):8711-87215 (1984), Townsend et al., U.S. Pat.No. 5,563,055); particle or microprojectile bombardment (U.S. Pat. No.5,100,792, EP-A-444882, EP-A-434616; Sanford et al, U.S. Pat. No.4,945,050; Tomes et al. (1995) “Direct DNA Transfer into Intact PlantCells via Microprojectile Bombardment”, in Plant Cell, Tissue and OrganCulture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag,Berlin); and McCabe et al. (1988) Biotechnology 6: 923-926);microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green etal. 91987) Plant Tissue and Cell Culture, Academic Press, Crossway etal. (1986) Biotechniques 4:320-334); electroporation (EP 290395, WO8706614, Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606;D'Halluin et al. 91992). Plant Cell 4:1495-1505) other forms of directDNA uptake (DE 4005152, WO 9012096, U.S. Pat. No. 4,684,611, Paszkowskiet al. (1984) EMBO J. 3:2717-2722); liposome-mediated DNA uptake (e.g.Freeman et al (1984) Plant Cell Physiol, 29:1353); or the vortexingmethod (e.g. Kindle (1990) Proc. Nat. Acad. Sci. USA 87:1228). Physicalmethods for the transformation of plant cells are reviewed in Oard(1991) Biotech. Adv. 9:1-11. See generally, Weissinger et al. (1988)Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Sciencesand Technology 5:27-37; Christou et al. (1988) Plant Physiol.87:671-674; McCabe et al. (1988) Bio/Technology 6:923-926; Finer andMcMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182; Singh et al.(1988) Theor. Appl. Genet. 96:319-324; Datta et al. (1990) Biotechnology8:736-740; Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85: 4305-4309;Klein et al. (1988) Biotechnology 6:559-563; Tomes, U.S. Pat. No.5,240,855; Buising et al. U.S. Pat. Nos. 5,322,783 and 5,324,646; Kleinet al. (1988) Plant Physiol 91: 440-444; Fromm et al (1990)Biotechnology 8:833-839; Hooykaas-Von Slogteren et al. 91984). Nature(London) 311:763-764; Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84:5345-5349; De Wet et al. (1985) in The Experimental Manipuation ofOvule Tissues ed. Chapman et al. (Longman, New York), pp. 197-209;Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al.(1992) Theor. Appl. Genet. 84:560-566; Li et al. (1993) Plant CellReports 12: 250-255 and Christou and Ford (1995) Annals of Botany 75:407-413; Osjoda et al. (1996) Nature Biotechnology 14:745-750, all ofwhich are herein incorporated by reference.

Agrobacterium transformation is widely used by those skilled in the artto transform dicotyledonous species. Recently, there has beensubstantial progress towards the routine production of stable, fertiletransgenic plants in almost all economically relevant monocot plants(Toriyama et al. (1988) Bio/Technology 6: 1072-1074; Zhang et al. (1988)Plant Cell rep. 7379-384; Zhang et al. (1988) Theor. Appl. Genet.76:835-840; Shimamoto et al. (1989) Nature 338:274-276; Datta et al.(1990) Bio/Technology 8: 736-740; Christou et al. (1991) Bio/Technology9:957-962; Peng et al (1991) International Rice Research Institute,Manila, Philippines, pp. 563-574; Cao et al. (1992) Plant Cell Rep. 11:585-591; Li et al. (1993) Plant Cell Rep. 12: 250-255; Rathore et al.(1993) Plant Mol. Biol. 21:871-884; Fromm et al (1990) Bio/Technology8:833-839; Gordon Kamm et al. (1990) Plant Cell 2:603-618; D'Halluin etal. (1992) Plant Cell 4:1495-1505; Walters et al. (1992) Plant Mol.Biol. 18:189-200; Koziel et al. (1993). Biotechnology 11194-200; Vasil,I. K. (1994) Plant Mol. Biol. 25:925-937; Weeks et al (1993) PlantPhysiol. 102:1077-1084; Somers et al. (1992) Bio/Technology10:1589-1594; WO 92/14828. In particular, Agrobacterium mediatedtransformation is now emerging also as an highly efficienttransformation method in monocots. (Hiei, et al. (1994) The PlantJournal 6:271-282). See also, Shimamoto, K. (1994) Current Opinion inBiotechnology 5:158-162; Vasil, et al. (1992) Bio/Technology 10:667-674;Vain, et al. (1995) Biotechnology Advances 13(4):653-671; Vasil, et al.(1996) Nature Biotechnology 14: 702).

Microprojectile bombardment, electroporation and direct DNA uptake arepreferred where Agrobacterium is inefficient or ineffective.Alternatively, a combination of different techniques may be employed toenhance the efficiency of the transformation process, e.g. bombardmentwith Agrobacterium-coated microparticles (EP-A-486234) ormicroprojectile bombardment to induce wounding followed byco-cultivation with Agrobacterium (EP-A-486233).

Plants which include a plant cell according to the invention are alsoprovided.

In addition to the regenerated plant, the present invention embraces allof the following: a clone of such a plant, seed, selfed of hybridprogeny and descendants (e.g. F1 and F2 descendants).

According to a further aspect of the invention there is provided anisolated nucleic acid molecule obtainable from Arabidopsis thalianawhich comprises a nucleic acid sequence encoding a polypeptide having

(1) GTase functionality; and(2) is capable of adding a glucosyl group via an O-glucosidic linkage toform

(a) a glucosyl ester of at least one of:

-   -   cinnamic acid; p-coumaric acid; caffeic acid; ferulic acid; and        sinapic acid; and/or

(b) a 4-O-glucoside of at least one of:

cinnamic acid; p-coumaric acid; caffeic acid; ferulic acid; sinapicacid; p-coumaryl aldehyde; coniferyl aldehyde; sinapyl aldehyde;p-coumaryl alcohol; coniferyl alcohol; and sinapyl alcohol.

In a further aspect of the invention there is provided a polypeptideencoded by an isolated nucleic acid molecule of the present inventionwherein the said polypeptide is selected from the polypeptides of FIGS.1B, 2B, 3B, 4B, 5B, 6B, 7B, 8B, 9B, 10B and 111B or functional variantsand/or parts thereof. Preferably the polypeptide is selected from thegroup of polypeptides of FIGS. 2B, 3B, 4B, 6B, 7B and 9B or functionalvariants and/or parts thereof. Preferably still the polypeptide isselected from the group of polypeptides selected from FIGS. 2B, 3B, 7Band 9B or functional variants and/or parts thereof. Most preferably thepolypeptide is one of the polypeptides shown in FIGS. 2B, 3B, 7B or 9B.Polypeptides encoded by the sense nucleic acid sequences presented inFIGS. 12-32 are also provided and readily derived from these sensesequences.

Variants of sequences having substantial identity or homology with theGTase molecules of the invention may be utilized in the practices of theinvention. That is, the GTase of FIGS. 1A-11A may be modified yet stillremain functional. Generally, the GTase will comprise at least about40%-60%, preferably about 60%-80%, more preferably about 80%-95%sequence identity with a GTase nucleotide sequence of FIGS. 1A-32herein.

The activity of functional variant polypeptides may be assessed bytransformation into a host capable of expressing the nucleic acid of theinvention. Methodology for such transformation is described in moredetail below.

In a further aspect of the invention there is disclosed a method ofproducing a derivative nucleic acid comprising the step of modifying anyof the sequences disclosed above, particularly the coding sequence ofFIGS. 1A, 2A, 3A, 4A, 5A, 6A, 7A, 8A, 9A, 10A, 11A, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32

Alternatively, changes to a sequence may produce a derivative by way ofone or more of addition, insertion, deletion or substitution of one ormore nucleotides in the nucleic acid, leading to the addition,insertion, deletion or substitution or one or more amino acids in theencoded polypeptide.

Other desirable mutations may be random or site directed mutagenesis inorder to alter the activity (e.g. specificity) or stability of theencoded polypeptide or to produce dominant negative variants which mayalter the flux through lignin biosynthetic pathways to alter the amountof lignin or an intermediate in the lignin biosynthetic pathway.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described with reference to the followingFigures and Examples which are not to be construed as limiting theinvention.

Scheme 1: The major intermediates in lignin biosynthesis pathway.

FIG. 1A: Sense nucleotide sequence of A062 (SEQ ID NO: 1). The codingregion starts from the first nucleotide and ends at the last nucleotide;

FIG. 1B: The amino acid sequence of A062 (SEQ ID NO: 2);

FIG. 1C: The antisense nucleotide sequence of A062 (SEQ ID NO:3);

FIG. 2A Sense nucleotide sequence of A320 (SEQ ID NO: 4). The codingregion starts from the first nucleotide and ends at the last nucleotide;

FIG. 2B The amino acid sequence of A320 (SEQ ID NO: 5);

FIG. 2C: The antisense nucleotide sequence of A320 (SEQ ID NO: 6);

FIG. 3A: Sense nucleotide sequence of A41 (SEQ ID NO: 7). The codingregion starts from the first nucleotide and ends at the last nucleotide;

FIG. 3B: The amino acid sequence of A41 (SEQ ID NO: 8);

FIG. 3C: The antisense nucleotide sequence of A41 (SEQ ID NO: 9);

FIG. 4A: Sense nucleotide sequence of A42 (SEQ ID NO: 10). The codingregion starts from the first nucleotide and ends at the last nucleotide;

FIG. 4B: The amino acid sequence of A42 (SEQ ID NO: 1);

FIG. 4C: The antisense nucleotide sequence of A42 (SEQ ID NO: 12);

FIG. 5A: Sense nucleotide sequence of A43 (SEQ ID NO: 13). The codingregion starts from the first nucleotide and ends at the last nucleotide;

FIG. 5B: The amino acid sequence of A43 (SEQ ID NO: 14);

FIG. 5C: The antisense nucleotide sequence of A43 (SEQ ID NO: 15);

FIG. 6A: Sense nucleotide sequence of A911 (SEQ ID NO: 16). The codingregion starts from the first nucleotide and ends at the last nucleotide;

FIG. 6B: The amino acid sequence of A911 (SEQ ID NO: 17);

FIG. 6C: The antisense nucleotide sequence of A911 (SEQ ID NO: 18);

FIG. 7A: Sense nucleotide sequence of A119 (SEQ ID NO: 19). The codingregion starts from the first nucleotide and ends at the last nucleotide;

FIG. 7B: The amino acid sequence of A119 (SEQ ID NO: 20);

FIG. 7C: The antisense nucleotide sequence of A119 (SEQ ID NO: 21);

FIG. 8A: Sense nucleotide sequence of A233 (SEQ ID NO: 22). The codingregion starts from the first nucleotide and ends at the last nucleotide;

FIG. 8B: The amino acid sequence of A233 (SEQ ID NO: 23);

FIG. 8C: The antisense nucleotide sequence of A233 (SEQ ID NO: 24);

FIG. 9A: Sense nucleotide sequence of A407 (SEQ ID NO: 25). The codingregion starts from the first nucleotide and ends at the last nucleotide;

FIG. 9B: The amino acid sequence of A407 (SEQ ID NO: 26);

FIG. 9C: The antisense nucleotide sequence of A407 (SEQ ID NO: 27);

FIG. 10A: Sense nucleotide sequence of A961 (SEQ ID NO: 28). The codingregion starts from the first nucleotide and ends at the last nucleotide;

FIG. 10B: The amino acid sequence of A961 (SEQ ID NO: 29);

FIG. 10C: The antisense nucleotide sequence of A961 (SEQ ID NO: 30);

FIG. 11A: Sense nucleotide sequence of A962 (SEQ ID NO: 31). The codingregion starts from the first nucleotide and ends at the last nucleotide;

FIG. 11B: The amino acid sequence of A962 (SEQ ID NO: 32);

FIG. 11C: The antisense nucleotide sequence of A962. (SEQ ID NO: 33);

FIG. 12: The sense nucleotide sequence of UGT71B5 (SEQ ID NO: 34);

FIG. 13 The sense nucleotide sequence of UGT71C3 (SEQ ID NO: 35);

FIG. 14 The sense nucleotide sequence of UGT71C5 (SEQ ID NO: 36);

FIG. 15 The sense nucleotide sequence of UGT71D1 (SEQ ID NO: 37);

FIG. 16 The sense nucleotide sequence of UGT73B1 (SEQ ID NO: 38);

FIG. 17 The sense nucleotide sequence of UGT73B2 (SEQ ID NO: 39);

FIG. 18 The sense nucleotide sequence of UGT73B4 (SEQ ID NO: 40);

FIG. 19 The sense nucleotide sequence of UGT73B5 (SEQ ID NO: 41);

FIG. 20 The sense nucleotide sequence of UGT73C1 (SEQ ID NO: 42);

FIG. 21 The sense nucleotide sequence of UGT731C (SEQ ID NO: 43);

FIG. 22 The sense nucleotide sequence of UGT73C5 (SEQ ID NO: 44);

FIG. 23 The sense nucleotide sequence of UGT73C6 (SEQ ID NO: 45);

FIG. 24 The sense nucleotide sequence of UGT73C7 (SEQ ID NO: 46);

FIG. 25 The sense nucleotide sequence of UGT74F2 (SEQ ID NO: 47);

FIG. 26 The sense nucleotide sequence of UGT76E1 (SEQ ID NO: 48);

FIG. 27 The sense nucleotide sequence of UGT76E11 (SEQ ID NO: 49);

FIG. 28 The sense nucleotide sequence of UGT76E12 (SEQ ID NO: 50);

FIG. 29 The sense nucleotide sequence of UGT76E2 (SEQ ID NO: 51);

FIG. 30 The sense nucleotide sequence of UGT78D1 (SEQ ID NO: 52);

FIG. 31 The sense nucleotide sequence of UGT89B1 (SEQ ID NO: 53);

FIG. 32 The sense nucleotide sequence of UGT72B3 (SEQ ID NO: 54);

FIG. 33 shows recombinant GST-UGT71C1 fusion protein purified from E.coli using glutathione-coupled Sepharose. The protein (5 μg) wasanalyzed using 10% SDS-PAGE and was visualized with Coomassive staining;

FIG. 34 shows three different glucose conjugates of caffeic acid,(caffeoyl-3-O-glucoside, caffeoyl-4-O-glucoside and1-O-caffeoylglucose), obtained from the glucosyltransferase reactionscontaining the recombinant UGT71C1, UGT73B3 and UGT84A1 respectively.Each assay contained 1-2 μg of recombinant UGT, 1 mM caffeic acid, 5 mMUDP-glucose, 1.4 mM 2-mercaptoethanol and 50 mM TRIS-HCl, pH 7.0. Themix was incubated at 30° C. for 30 min and was analyzed by reverse-phaseHPLC subsequently. Linear gradient (10-16%) of acetonitrile in H₂O at 1ml/min over 20 min was used to separate the glucose conjugates fromcaffeic acid.

FIG. 35A shows the pH optima of UGT71C1 glucosyltransferase activitymeasured over the range pH 5.5-8.0 in the reactions containing 50 mMbuffer, 1 μg of UGT71C1, 1 mM caffeic acid, 5 mM UDP-glucose and 1.4 mM2-mercaptoethanol. The mix was incubated at 30° C. for 30 min. Thereaction was stopped by the addition of 20 μl of trichloroacetic acid(240 mg/ml) and was analyzed by reverse-phase HPLC subsequently. Thespecific enzyme activity was expressed as nanomoles of caffeic acidglucosylated per second (nkat) by 1 mg of protein in 30 min of reactiontime at 30° C. FIG. 35B, the time course of UGT71C1 glucosyltransferaseactivity was studied by measuring the amount of caffeic acidglucosylated by 1 μg of UGT71C1 in 50 mM TRIS-HCll, pH 7.0. Thereactions were carried out and analyzed as described in A;

FIG. 36 shows UGT71C1 transgenic Arabidopsis thaliana plants and theirability to glucosylate caffeic acid; and

FIG. 37 summarises the GTase activities of various GTase polypeptideswith respect to various anti-oxidant substrates.

EXAMPLES Materials and Methods Transformation of Woody Plant Species

The transformation of woody plant species is known in the art. See U.S.Pat. No. 4,795,855 and WO9118094; EP1050209 and WO9725434. Each of thesepatents are incorporated in their entirety by reference.

Transformation of Non-Woody Plant Species

Methods used in the transformation of plant species other than woodyspecies are well known in the art and are extensively referenced herein.

Identification of GTase sequences

The GTase sequence identification was carried out using GCG software(Wisconsin package, version 8.1). Blasta programme was used to searchArabidopsis protein sequences containing a PSPG (plant secondary productUDP-glucose glucosyltransferase) signature motif (Hughes and Hughes(1994) DNA Sequence 5, 41-49) in EMBL and GenBank sequence database. Thedatabase information on the GTases described in the present inventionare listed in Table 1.

Amplification and Cloning of the GTase Sequences

The GTase sequences were amplified from Arabidopsis thaliana Columbiagenomic DNA with specific primers (Table 2), following standardmethodologies (Sambrook et al (1989) Molecular Cloning: A LaboratoryManual, 2^(nd) Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.). 50 ng of genomic DNA isolated from Arabidopsis thaliana Columbiawere incubated with 1× pfu PCR buffer (Stratagene), 250 μM dNTPS, 50pmole primer for each end, and 5 units of pfu DNA polymerase(Stratagene) in a total of 100 μl. The PCR reactions were carried out asoutlined in the programme described in Table 3.

After PCR amplification, the products were double digested byappropriate restriction enzymes listed in Table 2 (bold type). Thedigested DNA fragments were purified using an electro-eluction method(Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, 2^(nd)Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) and ligatedinto the corresponding cloning site on pGEX2T plasmid DNA (Pharmacia) byT4 DNA ligase (NEB) at 16° C. overnight. The resulting recombinantplasmid DNA was amplified in E. coli XL1-blue cells and was confirmedwith the restriction enzymes listed in Table 2 (bold type) following themethod described by Sambrook et al (1989) Molecular Cloning: ALaboratory Manual, 2^(nd) Ed., Cold Spring Habor Laboratory, Cold SpringHarbor, N.Y.).

Preparation of Glucosyltransferase Recombinant Proteins

E. coli cells carrying recombinant plasmid DNA as described above weregrown at 37° C. overnight on 2YT (16 g bacto tryptone, 10 g bacto-yeastextract, 5 g NaCl per litre) agar (1.8% w/v) plate which contained 50μg/ml ampicillin. A single colony was picked into 2 ml of 2YT containingthe same concentration of ampicillin. The bacterial culture wasincubated at 37° C. with moderate agitation for 6 h. The bacterialculture was transferred into 1 L of 2YT and incubated at 20° C.subsequently. 0.1 mM IPTG was added when the culture reached logarithmicgrowth phase (A 600 nm 0.5). The bacterial culture was incubated foranother 24 h. The cells were collected by a centrifugation at 7,000×gfor 5 min at 4° C. and resuspended in 5 ml spheroblast buffer (0.5 mMEDTA, 750 mM sucrose, 200 mM Tris, pH 8.0). Lysozyme solution was addedto a final concentration of 1 mg/ml. 7-fold volume of 0.5× spheroblastbuffer was poured into the suspension immediately and the suspension wasincubated for 4° C. for 30 min under gentle shaking. The spheroblastswere collected by a centrifugation at 12,000×g for 5 min at 4° C., andresuspended in 5 ml ice cold PBL buffer (140 mM NaCl, 80 mM, NA₂ HPO₄,15 mM KH₂PO₄). 2 mM of PMSF was added into suspension immediately andthe suspension was centrifuged at 12,000×g for 20 min at 4° C. in orderto remove the cell debris. After the centrifugation, the supernatant wastransferred to a 15 ml tube. 200 μl of 50% (v/v) slurry ofGlutathione-coupled Sepharose 4B were added into the tube and themixture was mixed gently for 30 min at room temperature. The mixture wasthen centrifuged at a very slow speed (500×g) for 1 min. the supernatantwas discarded. The beads were washed with 5 ml ice cold PBS buffer threetimes. After each wash, a short centrifugation was applied as describedabove to sediment the Sepharose beads. To recover the expressed proteinfrom Sepharose beads, 100 μl of 20 mM reduced glutathione were used toresuspend the beads. After 10 min incubation at room temperature, thebeads were collected and the supernatant containing the expressedprotein was collected. The elution step was repeated once, and bothsupernatant fractions were combined and stored at 4° C. for proteinassay and further studies.

Protein Concentration Assay

The protein assays were carried out by adding 10 μl of protein solutioninto 900 μl of distilled water and 200 μl of Bio-Rad Protein Assay Dye.The absorbance at 595 nm was read. A series of BSA (bovine seriumalbumin) at different concentration was used as standard. Regressionline was plotted based on the coordinates of the BSA concentrationagainst the reading at 595 nm. The concentration of protein sample wastherefore estimated from the regression line after the protein assay.

Assay for Enzyme Activity

A standard glucosylation reaction was set up by mixing 2 μg ofrecombinant proteins with 14 mM 2-mercaptoethanol, 5 mM UDP-glucose, 1mM of various lignin or antioxidant substrate, 100 mM Tris, pH 7.0, to atotal volume of 200 μl. The reaction was carried out at 30° C. for 30min and stopped by the addition of 20 μl trichloroacetic acid (240mg/ml). All the samples were stored at −20° C. before the liquidchromatographic assay.

High-Performance Liquid Chromatographic

Reverse-phase HPLC (Waters Separator 2690 and Waters Tunable AbsorbanceDetector 486, Waters Limited, Herts, UK) using a Columbus 5μ C₁₈ column(250×4.60 mm, Phenomenex). Linear gradient of acetonitrile in H₂O (allsolutions contained 0.1% trifluoroacetic acid) at 1 ml/min over 20 min,was used to separate the glucose conjugates from their aglycone. TheHPLC methods were described as the following: cinnamic acid, λ_(288 nm),10-55% acetonitrile; p-coumaric acid, λ_(311 nm, 10)-25% acetonitrile;caffeic acid, λ_(311 nm, 10)-16% acetonitrile; ferulic acid,λ_(311 nm, 10)-35% acetonitrile; sinapic acid, λ_(306 nm, 10)-40%acetonitrile; p-coumaryl aldehyde, λ_(315 nm, 10)-46% acetonitrile;coniferyl aldehyde, λ_(283 nm, 10)-47% acetonitrile; sinapyl aldehyde,λ_(280 nm, 10)-47% acetonitrile; p-coumaryl alcohol, p_(283 nm, 10)-27%acetonitrile; coniferyl alcohol, λ_(306 nm, 10)-25% acetonitrile;sinapyl alcohol, λ_(285 nm, 10)-25% acetonitrile. The retention time(R_(t)) of the glucose conjugates analysed is listed in the following:cinnamoylglucose, R_(t)=12.3 min; p-coumaroylglucose, R_(t)=10.6 min;caffeoylglucose, R_(t)=8.5 min; feruloylglucose, R_(t)=10.3 min;sinapoylglucose, R_(t)=9.7 min; caffeoyl-4-O-glucoside, R_(t)=6.8 min;feruloyl-4-O-glucoside, R_(t)=7.8 min; sinapoyl-4-O-glucoside, R_(t)=8.2min; coniferin, R_(t)=8.2 min; syringin, R_(t)=9.1 min.

The recombinant GTases were shown to have GTase activity towards themajor intermediates of the lignin biosynthesis pathway (Tables 5 and 6).It is clear from these results that the GTases display differentspecific activity reaction profiles relative to each other on thevarious lignin precursor substrates utilised. Michaelis-Menten kineticswere also studied on several of the GTases against their preferredsubstrates (Tables 7 and 8). It is clear from these results that theGTases display different enzyme kinetics for different substrates.

The results (in total) indicate that certain GTases show a greaterpotential for use in the alteration of lignin biosynthesis inplanta thanothers.

Reducing the Formation of Monolignol Glucosides In Planta

In one approach to reduce the formation of monolignol glucosides inplanta, A119 and A407 are down regulated using an antisense strategy(A). Expression of the A119 and A407 antisense sequences is driven bythe gene's own promoter. An alternative approach (B) is to modify theUDP-glucose binding motif through an in vitro mutagenesis method (Lim etal., 1998) such that the mutant protein is able to bind the monolignolsubstrates but loses its catalytic activity. Such mutant proteins arethought to compete with the functional native protein by bindingspecifically to monolignols, thereby reducing the formation ofmonolignol glucosides.

Anti-Sense Approach Amplification and Cloning of the A119 and A407Promoter Sequences

Approximately 2 kb of the 5′ flanking sequences of A119 and A407 areamplified directly from genomic DNA by PCR. The promoter fragments arethen cloned into a pBluescript plasmid vector (Sambrook et al., 1989).

Construct Chimaeric Genes of A119 and A407 Promoter and Their ORF Regionin Antisense Orientation.

The A119 and A407 cDNA fragments are amplified from pGEXA119 andpGEXA407 by PCR. The fragments are then ligated correspondingly into theA119 and A407 promoter constructs described in (A)-(1) with the ORFregion in the antisense orientation.

Preparation of Binary Construct Containing the A119 and A407 AntisenseChimaeric Gene

The DNA fragments containing the A119 and A407 antisense chimaeric genesare amplified by PCR from the chimaeric constructs described in (A)-(2).The fragments are then ligated into a binary vector (Sambrook et al.,1989). The final constructs are transformed into plants subsequently.

Mutant Gene Approach In Vitro Site Mutagenesis to Modify the UDPglucoseBinding Motif in A119 and A407

In vitro site mutagenesis is carried by PCR to modify the sequencesencoding the UDPglucose binding motif in A119 and A407 (Lim et al.,1998). The constructs pGEXA119 and pGEXA407 are used in the DNAtemplates in the PCR reaction.

Construct Chimaeric Mutant Genes Regulated by A119 and A407 Promoters

The A119 and A407 mutant genes are amplified from the pGEXA119 andpGEXA407 mutant constructs described in (B)-(1) by PCR. The A119 andA407 mutant gene fragments are then ligated into the A119 and A407promoter constructs described in (A)-(1) with the ORF region in thesense orientation.

Preparation of Binary Construct Containing the Chimaeric Mutant GenesA119 and A407

The DNA fragments containing the A119 and A407 mutant chimaeric genesare amplified by PCR from the chimaeric constructs described in (B)-(2).The fragments are then ligated into a binary vector (Sambrook et al.,1989). The final constructs are transformed into plants subsequently.

Enhancing the Formation of Monolignol Glucosides in Planta

The CaMV 35S promoter fragment is used to drive the expression of A119and A497. DNA fragments containing A119 and A407 ORF sequences areamplified from pGEXA119 and pGEXA407 correspondingly by PCR. The DNAfragments are ligated downstream of the CaMV 35S promoter. Theconstructs are used to transform plants such that the lignin content andcomposition is altered.

TABLE 1 Database information on eleven Arabidopsis GTase genes GeneDatabase BAC/P1 gene name in name protein_id chromosome acc. no. clonedatabase A062 Gi|3935156 I ac005106 T25N20 T25N20.20 A320 Not annotatedIII ab019232 MIL23 not annotated A41 Emb|CAB10326.1 IV z97339 FCA4d13780c A42 Emb|CAB10327.1 IV z97339 FCA4 d13785c A43 Emb|CAB10328.1 IVz97339 FCA4 d13790c A911 Gi|2642451 II ac002391 T20D16 T20D16.11 A119Not annotated V ab018119 MSN2 not annotated A233 Wrongly annotated IVal021961 F28A23 wrongly annotated A407 Gi|3319344 V af077407 F9D12F9D12.4 A961 Gi|3582329 II ac005496 T27A16 T27A16.15 A962 Gi|3582341 IIac005496 T27A16 T27A16.16

Parameters used for the search of the above Arabidopsis sequences andthe programme used are as follows:

NETBLAST with the default settings:

Infile2=nr Matrix=Blosum 62 Translate=1 Dbtranslate=1

TABLE 2 DNA sequences and restriction enzyme sites in primers used inamplification of 11 Arabidopsis Gtase sequences from genomic DNA.Sequence complementary to either end of the ORFs are under- lined.Restriction enzyme sites that were used in making expression constructswere in BOLD type. restriction primer DNA sequence (5′→3′) enzyme sitesA062 5′ CGGGTGATCAGGTACCATGGCGCCACCGCATTTTC BclI and KpnI SEQ ID NO: 55A062 3′ CGGAATTCGTCGAGTTACTTTACTTTTACCTCCTC EcoRI and SalI SEQ. ID NO:56 A320 5′ CCCCCGGGTACCATGGAGCTAGAATCTTCTCTCC SmaI and KpnI SEQ. ID NO:57 A320 3′ CGGAATTCTCGAGTTAAAAGGTTTTGATTGATCC EcoRI and XhoI SEQ. ID NO:58 A41 5′ TGGGATCCATATCAGAAATGGTGTTC BamHI SEQ. ID NO: 59 A41 3′GGGAATTCCTAGTATCCATTATCTTTAGTC EcoRI SEQ. ID NO: 60 A42 5′GGGGATCCATGGACCCGTCTCGTCATACTC BamHI SEQ. ID NO: 61 A42 3′GGGAATTCCACTAGTGTTCTCCGTTGTCTTC EcoRI SEQ. ID NO: 62 A43 5′GGGGATCCAATATGGAGATGGAATCGTCGTTAC BamHI SEQ. ID NO: 63 A43 3′GGGAATTCCTTACACGACATTATTAATGTTTG EcoRI SEQ. ID NO: 64 A911 5′GGGGTACCTGATCAATAATGGGCAGTAGTGAGGG KpnI and BclI SEQ. ID NO: 65 A911 3′CGGAATTCGTCGACGAGTTAGGCGATTGTGATATC EcoRI and SalI SEQ. ID NO: 66 A1195′ CGGGATCCGGTACCATGCATATCACAAAACCACAC BamHI and KpnI SEQ. ID NO: 67A119 3′ CGGAATTCGCTAGCTAAGCACCACGTGACAAGTCC EcoRI and NheI SEQ. ID NO:68 A233 5′ CGGGATCCGGTACCATGAGTAGTGATCCTCATCGT BamHI and KpnI SEQ. IDNO: 69 A233 3′ CGGGATCCGAATTCTAGGAGGTAAACTCTTCTATG BamHI and SEQ. ID NO:70 EcoRI A407 5′ CGGGATCCGGTACCATGCATATCACAAAACCACAC BamHI and KpnI SEQ.ID NO: 71 A407 3′ CGGAATTCGTCGACCTAAGCACCACGTCCCAAG EcoRI and SalI SEQ.ID NO: 72 A961 5′ GGGTGATCAGGTACCATGGGGAAGCAAGAAGATG BclI and KpnI SEQ.ID NO: 73 A961 3′ CGGAATTCGTCGACTACTTACTTATAGAAACGCCG EcoRI and SalISEQ. ID NO: 74 A962 5′ GAAGATCTGGTACCATGGCGAAGCAGCAAGAAG BglII and KpnISEQ. ID NO: 75 A962 3′ CGGAATTCGTCGACCGATGAAAGCCCATCTATG EcoRI and SalISEQ. ID NO: 76

TABLE 3 PCR programme Stage I (1 cycle) Stage II (40 cycles) Stage III(1 cycle) 95° C. 5 min 95° C. 1 min 95° C. 2 min 55° C. 2 min 55° C. 1min 55° C. 2 min 72° C. 3 min 72° C. 2 min 72° C. 5 min

TABLE 4 The HPLC conditions Detector Acetonitrile Wavelength LigninPrecursors Gradient (%) (nm) cinnamic acid 10-55 288 p-coumaric acid10-25 311 caffeic acid 10-16 311 ferulic acid 10-35 311 sinapic acid10-40 306 p-coumaryl aldehyde 10-46 315 coniferyl aldehyde 10-47 283sinapyl aldehyde 10-47 280 p-coumaryl alcohol 10-27 283 coniferylalcohol 10-25 306 sinapyl alcohol 10-25 285

TABLE 5 Specific activity of the recombinant GTases producing glucoseester against lignin precursors Each assay contained 0.5 m□ of potentialsubstrates, 5 mM UDPG and 0.2 μg of recombinant GTases in a total volumeof 200 μl. The reactions were incubated at 20° C. for 30 min and werestopped by addition of 20 μl TCA (240 mg/ml). Each reaction mix was thenanalysed using HPLC. The specific activity (nkat/mg) of the recombinantGTase is defined as the amount of substrate (nmole) converted to glucoseester per second by 1 mg of protein at 20° C. under the reactionconditions. A41 A320 A42 A43 A911 A062 Cinnamic acid 0.30 0.06 14.210.02 8.77 1.62 p-coumaric acid 13.53 0.05 4.69 0.03 4.31 2.54 Caffeicacid 2.61 0.05 0.62 0.01 0.77 0.26 Ferulic acid 6.64 0.54 15.63 0.042.88 0.08 Sinapic acid 5.35 15.58 11.97 0.05 0.15 0.1

TABLE 6 Specific activity of the recombinant GTases producingO-glucosides against lignin precursors The reactions were set upfollowing the conditions described in Table 1. All the reactions, exceptthose containing the aldehydes, were stopped by the addition of TCA. Thealdehyde assay mixs were injected into HPLC immediately after thereactions were completed. The specific activity (nkat/mg) of therecombinant GTase is defined as the amount of substrate (nmole)converted to 4-O-glucoside per second by 1 mg of protein at 30° C. underthe reaction conditions. A233 A119 A407 A961 A962 Cinnamic acid ND^(a)ND ND ND ND p-coumaric acid 0.09 0.02 0.01 0.01 0.01 caffeic acid 0.480.13 0.07 0.07 ND ferulic acid 0.37 14.48  0.25 ND ND sinapic acid 0.39102.56  65.39  0.01 0.01 p-coumaryl aldehyde ND 0.03 ND 0.01 0.02Coniferyl aldehyde ND 1.08 ND 0.16 0.34 sinapyl aldehyde ND 4.55 ND 0.570.50 p-coumaryl alcohol ND ND ND ND ND Coniferyl alcohol 0.46 67.53 2.78 0.57 0.49 sinapyl alcohol 0.05 126.16  114.76  0.35 0.45 ^(a)ND,not detected

TABLE 7 Kinetic studies on the recombinant GTases producing glucoseesters against lignin precursors A41 A320 A42 A911 A062 K_(m) V_(max)K_(m) V_(max) K_(m) V_(max) V_(max) V_(max) MM nkat/mg mM nkat/mg mMnkat/mg K_(m) mM nkat/mg K_(m) mM nkat/mg 1.51 — 1.80 — 0.72 — 1.05 —2.36 — — — — — 0.49 19.42 0.05 9.06 4.33 2.87 0.10 16.13 — — 0.40  6.670.39 11.10  5.05 4.91 0.06 20.24 — — 0.20  1.67 0.23 1.18 — — 0.35 11.35— — 0.36 18.35 0.34 6.91 — — 0.24  6.78 0.06 8.37 0.13 12.80 — — — —

TABLE 8 Kinetic studies on the recombinant GTases producing O-glucosidesagainst lignin precursors A119 A407 K_(m) V_(max) K_(m) V_(max) mMnkat/mg mM nkat/mg UDPG 0.93 — 0.89 — ferulic acid 0.25  18.87 — —sinapic acid 0.51 131.58 0.14  75.19 coniferyl 0.26  92.59 — — alcoholsinapyl alcohol 1.10 322.58 1.07 357.10

TABLE 9 ¹H and ¹³C NMR spectra were recorded in deuterated methanol at500 MHz and 125 MHz respectively. Chemical shifts are given on δ scalewith TMS as internal standard. The position on the aromatic ring beginswith the carbon joining the propanoic acid. d, doublet; dd, doublet ofdoublets; m, multiplet; J, coupling constant. Caffeic acidCaffeoyl-3-O-glucoside Position δ_(H) δ_(C) δ_(H) δ_(C) C1 — 128.1 —127.6 C2 7.02 (1H, d, J = 2.0 Hz) 115.2 7.47 (1H, d, J = 2.0 Hz) 117.0C3 — 146.7 — 146.0 C4 — 149.4 — 150.6 C5 6.77 (1H, d, J = 8.0 Hz) 116.66.84 (1H, d, J = 8.5 Hz) 117.8 C6 6.92 (1H, dd, J = 8.0, 2.0 Hz) 122.87.13 (1H, dd, J = 8.5, 2.0 Hz) 125.6 C7 7.53 (1H, d, J = 16.0 Hz) 146.97.45 (1H, d, J = 14.5 Hz) 146.6 C8 6.21 (1H, d, J = 15.5 Hz) 116.3 6.33(1H, d, J = 14.5 Hz) 116.1 C9 — 171.5 — 170.4 Glc-1 ~4.86 (signalinterrupted) 103.9 Glc-2 74.5 Glc-3 78.0 Glc-4 {close oversize brace}3.40-3.50 (4H, m) 71.0 Glc-5 77.2 Glc-6 3.93 (1H, dd, J = 12.0, 2.0 Hz)62.4 3.71 (1H, dd, J = 12.0, 5.5 Hz)

TABLE 10 Each assay contained 1 □g of UGT71C1, 1 mM phenolic compound, 5mM UDP-glucose, 1.4 mM 2-mercaptoethanol and 50 mM TRIS-HCl, pH 7.0. Themix was incubated at 30° C. for 30 min. The reaction was stopped by theaddition of 20 □l of trichloroacetic acid (240 mg/ml) and was analysedby reverse-phase HPLC subsequently. The results represent the mean ofthree replicates ± standard deviation. Specific activity Substratenkat/mg o-Coumaric acid 1.5 ± 0.2 m-Coumaric acid 1.2 ± 0.2 p-Coumaricacid 0 Caffeic acid 2.9 ± 0.8 Ferulic acid 0 Sinapic acid 0 Esculetin34.8 ± 4.2  Scopoletin 29.4 ± 3.9  Salicylic acid 0 4-hydroxybenzoicacid 0 3,4-dihydroxybenzoic acid 0 Eriodictyol 0 Luteolin 0.7 ± 0.1Quercetin 1.4 ± 0.4 Catechin 0 Cyanidin 0

1. A transgenic plant comprising a nucleic acid molecule which encodes apolypeptide which has glucosyltransferase activity and is encoded by i)a nucleic acid molecule selected from the group consisting of SEQ IDNOs: 1, 4, 7, 10, 13, 16, 22, 25, 28, 31, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 and 54; or ii) anucleic acid molecule which hybridizes to SEQ ID NO: 19 or any of thesequences represented in (i) above and which modifies biosyntheticintermediates in lignin polymerization; or iii) a nucleic acid moleculewhich is degenerate as a result of the genetic code to SEQ ID NO: 19 orany of the sequences defined in (i) and (ii) above.
 2. A transgenicplant according to claim 1, wherein said intermediates are selected fromthe group consisting of: cinnamic acid; p-coumaric acid; caffeic acid;ferulic acid; sinapic acid; p-coumaryl aldehyde; coniferyl aldehyde;sinapyl aldehyde; p-coumaryl alcohol; coniferyl alcohol and sinapylalcohol.
 3. A transgenic plant according to claim 1, wherein the nucleicacid molecule anneals under stringent hybridization conditions to anucleic acid comprising a sequence selected from the group consisting ofSEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 and
 54. 4. Atransgenic plant according to claim 1, wherein the nucleic acid moleculeis SEQ ID NO: 25, and the intermediates are monolignol glucosides.
 5. Atransgenic plant comprising a nucleic acid molecule, wherein the nucleicacid molecule is selected from the group consisting of: i) antisensesequences selected from the group consisting of SEQ ID NOs: 3, 6, 9, 12,15, 18, 21, 24, 27, 30, parts thereof, and antisense sequence of thesense sequences presented in SEQ ID NOS: 34-54; and ii) antisensesequences which hybridize to any of the sense sequences according toclaim 1 and which inhibit GTase activity.
 6. A transgenic plantaccording to claim 5, wherein the antisense sequence hybridizes to asequence selected from the group consisting of SEQ ID NOS: 19 and
 25. 7.A transgenic plant according to claim 1, wherein the nucleic acid iscDNA.
 8. A transgenic plant according to claim 1, wherein the nucleicacid is genomic DNA.
 9. A transgenic plant according to claim 1, whereinthe plant is a woody plant selected from the group consisting of poplar;eucalyptus; Douglas fir; pine; walnut; ash; birch; oak; teak and spruce.10. A transgenic plant according to claim 9, wherein said plant ispoplar.
 11. A transgenic plant according to claim 1, wherein the plantis a non-woody plant species.
 12. A method for the manufacture of paperor paperboard comprising: i) pulping transgenic wood material derivedfrom a transgenic woody plant according to claim 8; and ii) producingpaper or paperboard from the pulped transgenic wood material.
 13. Atransgenic eukaryotic cell comprising a nucleic acid molecule whichencodes a polypeptide which has glucosyltransferase activity; saidnucleic acid molecule being selected from the group consisting of: i)nucleic acid molecules comprising a sequence selected from the groupconsisting of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53 and 54; ii) nucleic acid molecules which hybridize to any of thesequences represented in (i) above and which glucosylate ananti-oxidant; and iii) nucleic acid molecules which are degenerate as aresult of the genetic code to any of the sequences defined in (i) and(ii) above.
 14. A transgenic eukaryotic cell according to claim 13,wherein the nucleic acid molecule is selected from the group consistingof SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 and 54.15. A transgenic eukaryotic cell according to claim 14, wherein thenucleic acid molecule is selected from the group consisting of SEQ IDNOs: 1, 7, 10, 13, 19, 22, 25 and
 28. 16. A transgenic prokaryotic cellcomprising a nucleic acid molecule which encodes a polypeptide which hasglucosyltransferase activity and is encoded by: i) a nucleic acidmolecule selected from the group consisting of SEQ ID NOs: 1, 4, 7, 10,13, 16, 19, 22, 25, 28, 31, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53 and 54; or ii) a nucleic acidmolecule which hybridizes to any of the sequences represented in (i)above and which glucosylates an anti-oxidant; or iii) a nucleic acidmolecule which is degenerate as a result of the genetic code to any ofthe sequences defined in (ii) and (iii) above.
 17. A transgenicprokaryotic cell according to claim 16, wherein the nucleic acidmolecule is selected from the group consisting of SEQ ID NOs: 1, 4, 7,10, 13, 16, 19, 22, 25, 28, 31, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53 and
 54. 18. A transgenicprokaryotic cell according to claim 17, wherein the nucleic acidmolecule is selected from the group consisting of SEQ ID NOs: 1, 7, 10,13, 19, 22, 25 and 28.